WO2003051331A1

WO2003051331A1 - Methods for treating wounds using melanin and related substances

Info

Publication number: WO2003051331A1
Application number: PCT/US2002/034564
Authority: WO
Inventors: Steven A. Baranowitz
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-10-26
Filing date: 2002-10-28
Publication date: 2003-06-26
Anticipated expiration: 2004-04-26
Also published as: AU2002353907A1

Abstract

The present relates to the prophylaxis and treatment of wounds such as decubitus ulcers and burns by the administration of an active substance which causes an increased concentration of melanin, melanin precursor, melanin derivatives, melanin variants and melanin analogs in the effected tissue. Such active substances include melanin, melanin precursor, melanin derivatives, melanin variants, melanin analogs, the enzyme tyrosinase, which catalyzes the reaction wherein naturally occurring melanin precursors are converted to melanin, tyrosinase gene, melanin-concentrating hormone and combinations thereof.

Description

METHODS FOR TREATING WOUNDS USING MELANIN AND RELATED SUBSTANCES

This application claims priority under 35 U.S.C. § 119(e) from Provisional Application No. 60/347,942, filed October 26, 2001.

FIELD OF THE INVENTION

The present invention relates to the prophylaxis and treatment of wounds such as decubitus ulcers (bed sores) and burns by the administration of an active substance which causes an increased concentration of melanin, melanin precursors, melanin derivatives, melanin variants and melanin analogs in the effected tissue. Such active substances include melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, the enzyme tyrosinase, which catalyzes the reaction wherein naturally occurring melanin precursors are converted to melanin, tyrosinase gene, melanin- concentrating hormone and combinations thereof.

BACKGROUND OF THE INVENTION

Melanin is a pigment which is found in many cells and organisms (Majerus, M.E.N., Melanism, New York: Oxford University Press 1998). It is resistant to analysis and degradation by chemical and physical agents (Prota,G. , Melanins And Melanogenesis, New York: Academic Press, 1992). Therefore the complete structures of many melanins remain unknown, although a few have been identified and even synthesized (Prota,G. , Melanins And Melanogenesis , New York: Academic Press, 1992).

Melanin is naturally found in small packets called melanosomes (Prota,G., Melanins And Melanogenesis, New York: Academic Press, 1992), which are membrane- enclosed vesicles. Melanin is a hard substance (Majerus, M.E.N. , Melanism, New York: Oxford University Press, 1998). The presence of many small melanosomes strengthens cells. The melanin in cells is separated by membranes and other cytoplasmic components, This arrangement permits flexibility in movement while preserving hardness to resist pressure.

Melanin is normally present in mammalian skin in cells called melanocytes (Prota,G. , Melanins And Melanogenesis , New York: Academic Press, 1992). Melanocytes are most numerous in the epidermis and dermis of the skin, but are present in other tissues such as the eye, some nerves, the brain, and some blood cells. In the epidermis there is approximately 1 melanocyte to 36 keratinocytes. In the dermis the ratio of melanocytes to fibroblasts is much more variable. Melanin is often found in healed skin (Majerus, M.E.N., Melanism, New York: Oxford University Press, 1998).

Melanin is regarded by some investigators as primarily a waste product of metabolism.

Mammalian colors are determined chiefly by two types, eumelanins and phaeomelanins. Eumelanins are derived from the precursor tyrosine and are generally insoluble and black or brown in color. Phaeomelanins have as their precursors tyrosine and cysteine and are generally alkali-soluble and lighter in color. Allomelanins ("allo" meaning other) are formed from nitrogen-free precursors, primarily catechol and 1,8- dihydroxynaphthalene (see The Merck Index, Tenth Edition, page 827, item 5629, Melanins). Quinones are the usual intermediates in allomelanin synthesis. The synthesis of melanins occurs in nature as well as being produced synthetically. A further group of low molecular weight yellow, red and violet pigments is known as trichochromes. The trichochromes are usually classified with the melanins, since they serve as pigments and are derived from the oxidation of tyrosine. The enzyme, tyrosinase, plays a key role in the synthesis of melanin and its derivatives. In mammals, tyrosinase is a glycosylated enzyme found in melanocytes.

It has been theorized that tyrosinase functions by means of separate catalytic sites; one site for tyrosinase hydroxylase activity, another site for dopa oxidase activity, and a third independent site for dopa as a cofactor. Hearing, V. J. et al., Biochem. J., 157 549 (1976). Tyrosinase may also play a role in catalyzing the oxidation of 5,6-dihydroxyindole to indole-5,6-quinone. Korner, A. M. et al. , Science 217, 1163 (1982). In vivo, mammalian tyrosinase undergoes extensive modification. When initially synthesized, tyrosinase has an apparent molecular weight of about 55,000. Glycosylation of the enzyme occurs as it is transferred through the Golgi complex and delivered to the melanocytes. Imokawa, G. et al. , J. Invest. Derm., 85, 165 (1985). During this modification of tyrosinase, sialic acid and 4 mol of asparagine-linked carbohydrate chains (containing mannose, glucosamine, galactose and fucose) are added to each mole of tyrosinase. Ferrini, V. et al. , Int. J. Biochem. 19, 229 (1987). The glycosylated tyrosinase has an apparent molecular weight of about 70,000. Laskin, J. D. et al., J. Biol. Chem. 261, 16626 (1986).

The glycosylated tyrosinase is delivered to the melanocytes by coated vesicles. In the melanocytes, the tyrosinase is membrane bound and aggregates into a high molecular weight form. In vivo, tyrosinase is under active metabolic control involving an active degradation system which results in a biological half-like of about ten hours. Jimenez, M. et al., Fed. Proc. Fedn. Am. Socs. Exp. Biol. 45, 1714 (1986).

SUMMARY OF THE INVENTION

The present invention is directed to therapeutic uses of melanin, melanin precursors, melanin derivatives, melanin analogs and related substances. One particular aspect of the invention relates to the treatment or prevention of wounds by the administration of active substances which cause an increased melanin concentration in the patient's wounds. Such substances include melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, melanin-concentrating hormone (MCH), tyrosinase, tyrosinase gene and combinations thereof.

In one aspect, the present invention a method for treating a wound in a mammal comprising administering to a mammal in need of such treatment an amount effective for treating said wound of an agent which causes an increased concentration of melanin and related substance in said wound.

In another aspect, the present invention provides a method for preventing a wound in a mammal comprising administering to a mammal in need of such treatment an amount of an agent effective to prevent said wound of an agent which causes an increased concentration of melanin and related substances in said wound.

In yet another aspect the present invention provides a wound healing device selected from a patch, a strip, a wound dressing or a band comprising an amount of melanin or related substance effective to treat a wound in a mammal. In yet another aspect the present invention provides a pharmaceutical formulation for treating wounds in a mammal comprising mineral oil and a wound healing effective amount of an agent which causes an increase in the concentration of melanin in said wound.

In a further aspect, the present invention provides a pharmaceutical formulation for treating wounds in a mammal comprising a topical antibacterial cream or ointment and a wound healing effective amount of an agent which causes an increased concentration of melanin and related substances in said wound.

In a still further aspect, the present invention provides a method for treating wounds in a mammal comprising administering to a mammal in need of such treatment an amount effective to treat said wound of a pharmaceutical formulation of claim 14 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

In a still further aspect, the present invention provides a pharmaceutical formulation for treating wounds in a mammal comprising an emollient selected from ammonium lactate, ichthammol ointment and lanolin and a wound healing effective amount of an agent which causes an increased concentration of melanin and related substances in said wound.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present description and claims.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety.

The present invention is useful for healing of wounds in a mammal having such injury by administering an amount effective to treat the wounds of an active substance which causes an increased concentration of melanin in the wound to aid in healing. Melanin, a melanin precursor, a melanin analog, a melanin variant or a melanin derivative can be administered to accomplish this result. Alternatively, the melanin necessary to aid wound healing may be concentrated in the wound by administration of melanin concentration hormone (MCH), or may be produced in the patient's body by administering tyrosinase which catalyzes the syntheses of naturally occurring melanin precursors to melanin. Furthermore, the administration of the tyrosinase gene causes the production of tyrosinase in the patient's body, thereby catalyzing the conversion of the naturally occurring melanin precursors to melanin. The present invention is further useful in protecting a mammal from developing a wound, such as a decubitus ulcer, by administering an amount effective to prevent the wound of melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, MCH, tyrosinase, tyrosinase gene or a combination thereof.

For the purposes of the present description, melanins are defined below and are further described and classified in the book entitled "Melanins," by R. A. Nicolaus, published in 1968 by Hermann, 115, Boulevard Saint-Germain, Paris, France. As defined by Nicolaus, melanins constitute a class of pigments which are widespread in the animal and vegetable kingdoms. While the name "melanin" in Greek means black, not all melanins as pigments are black but may vary from brown to yellow. The following are some characteristics of melanin that makes it an excellent substance to prevent and treat wounds. a. It is hard (Majerus, M.E.N., Melanism, New York: Oxford University Press, 1998); b. It resists abrasion (Majerus, M.E.N., Melanism, New York: Oxford University Press, 1998); c. It is anti-inflammatory (Avramidis, N. et al. , Anti-Inflammatory And

Immunomodulating Properties Of Grape Melanin, Arzneim. -For sch ./Drug Res. 48:764- 771, 1998; Mohagheghpour, N. , et al., Synthetic Melanin Suppresses Production Of Proinflammatory Cytokines, Cellular Immunology 199:25-36, 2000) d. It is anti-infectious, including anti-viral (Sidibe S. et al., Effects of serotonin and melanin on in vitro HIV-1 infection, J Biol Regul Homeost Agents, 10: 19-24,

1996); e. It absorbs toxins (Nordlund, J.J. et al., The Pigmentary System, Oxford University Press, New York, 1998); f. It absorbs ultraviolet and other forms of radiation (Prota.G. , Melanins And Melanogenesis , New York: Academic Press, 1992); g. It may control protein synthesis by inhibiting DNA polymerase (Eckhart, L., et al., Melanin Binds Reversibly To Thermostable DNA Polymerase And Inhibits Its Activity. Biochemical And Biophysical Research Communications 271:726- 730, 2000 Goss, R. J. , Principles of Regeneration, Academic Press, New York, 1969);

h. It protects DNA. (Nordlund, J.J. et al., The Pigmentary System, Oxford University Press, New York, 1998); i. Melanin also has certain semiconductor like electrical properties which may facilitate cell metabolism (Prota,G., Melanins And Melanogenesis, New York: Academic Press, 1992); j. It is a free radical scavenger (Nordlund, J.J. et al. , The Pigmentary

System, Oxford University Press, New York, 1998); k. It is anti-oxidant (Nordlund, J.J. et al., The Pigmentary System,

Oxford University Press, New York, 1998); 1. It has been suggested that melanin can inhibit certain enzymes

(Eckhart, L. et al., Melanin Binds Reversibly To Thermostable DNA Polymerase And

Inhibits Its Activity. Biochemical And Biophysical Research Communications 271:726- 730, 2000) such as those which are released by lysosomes during injury and cause further tissue breakdown; m. It can bind proteins (Nordlund, J.J. et al. , The Pigmentary System,

Oxford University Press, New York, 1998); n. It has also been suggested that melanin minimizes cell mediated immunological reactions, thus again having anti-inflammatory effects (Mohagheghpour, N., et al. , Synthetic Melanin Suppresses Production Of Proinflammatory Cytokines, Cellular

Immunology 199:25-36, 2000); o. Melanin absorbs oxygen (Nordlund, J.J. et al. , The Pigmentary System, Oxford University Press, New York, 1998), and it is said that an anaerobic environment stimulates tissue regeneration (Goss, R. J. , Principles of Regeneration,

Academic Press, New York, 1969); p. Other properties of melanin that may be beneficial to healing wounds include the ion-exchange modulating properties of melanin (Nordlund, J.J. et al., The Pigmentary System, Oxford University Press, New York, 1998); q. Because melanin is dark, it absorbs heat more rapidly that light surfaces (Majerus, M.E.N., Melanism, New York: Oxford University Press, 1998) . Thus whether one discusses absorbing heat from the ambient environment, or from ultraviolet light or any other heat source, the presence of melanin enables more rapid heating of an area by any heat source. Since a one degree Celsius increase in skin temperature speeds skin, or other tissue wound healing by a factor of 2 or more (according to many studies) the melanin containing wound healing device, e.g., a melanin impregnated patch, when placed in the sun or next to a heat source, would also absorb heat and stimulate healing of the wound; r. The presence of melanin in discrete beads or melanosomes, interspersed among softer material, permits transport of other cellular components around melanosomes. Thus it is a porous barrier; s. Melanin is paramagnetic (Nordlund, J.J. et al., The Pigmentary

System, Oxford University Press, New York, 1998); t. Without wishing to be bound by theory, it is believed that melanin can be used as a substrate to enhance migration of cells into a location. Definitions

In order to provide a clear and consistent understanding of the specification and claims, including the scope given to such terms, the following definitions are provided: Wound: A wound is defined as a breach of the structural integrity, or impairment of the physiological function, of a tissue or organ. The structural integrity can be broken by being pierced, cut, burnt, or otherwise injured. Non-limiting examples of wounding of physiological function include impairment of the barrier functions with respect to heat, cold, water loss, and microbial invasion.

Administration: The application or delivery of a drug to a mammal in need of the drug. This term is intended to include any means of administration which accomplishes the application or delivery of the drug (i.e. , topical, oral, aerosol, suppository, parenteral, e.g., intravenous, intramuscular, subcutaneous injection, e.g. , into the tissue, peritonealy and the like). The term is also intended to include any means necessary to accomplish such administration. The term is further intended to include the in vivo production of a drug or aggregation of a drug moderated by another substance such as an enzyme (tyrosinase) or enzyme gene (tyrosinase gene) to moderate production of a drug (melanin) or its precursors, or a concentrating hormone (MCH) subcutaneously to moderate drug (melanin) concentration.

Treatment: Treatment is defined as administration to a mammal suffering from wounds or burns or administration to a mammal at risk for such wounds, e.g. , bedridden patients who are at risk for developing decubitis ulcers or bed sores.

Melanin: Melanins are polymers produced by polymerization of reactive intermediates. The polymerization mechanisms include but are not limited to autoxidation, enzyme catalyzed polymerization and free radial initiated polymerization. The reactive intermediates are produced chemically or enzymatically from precursors. Suitable enzymes include, but are not limited to peroxidases and catalases, polyphenol oxidases, tyrosinases, tyrosine hydroxylases or lacases. The precursors which are connected to the reactive intermediates are hydroxylated aromatic compounds. Suitable hydroxylated aromatic compounds include, but are not limited to 1) phenols, polyphenols, aminophenols and thiophenols of aromatic or polycyclicaromatic hydrocarbons, including but not limited to phenol, tyrosine, pyrogallol, 3-aminotyrosine, thiophenol and . alpha. -naphthol; 2) phenols, polyphenols, aminophenols, and thiophenols of aromatic heterocyclic or heteropoly cyclic hydrocarbons such as but not limited to 2-hydroxypyrrole,4-hydroxy-l,2-pyrazole, 4- hydro xypyridine, 8-hydroxyquinoline, and 4,5-dihydroxybenzothiazole. The term melanin includes naturally occurring melanins which are usually high molecular weight polymers (generally, molecular weights in the millions) and low molecular weight polymers as well as melanin analogs as defined below. Naturally occurring melanin includes eumelanins, phaeomelanins, neuromelanins and allomelanins. The term melanin is also intended to include trichochromes when used hereafter. The term "melanin" is further intended to include both melanin, melanin precursors, melanin analogs, melanin variants and melanin derivatives unless the context dictates otherwise. Melanin Analog: Melanin in which a structural feature that occurs in naturally occurring or enzymatically produced melanins is replaced by an unusual substituent divergent from substituents traditionally present in melanin. An example of an unusual substituent is selinium in place of sulfur, such as selinocysteine.

Melanin Derivative: This term is intended to include any derivative of melanin which is capable of being converted to either melanin or a substance having melanin activity. An example of a melanin derivative is melanin attached to a dihydrotrigonelline carrier such as described in Bodor, N., Ann. N.Y. Acad. Sci. 507, 289 (1987) to enable the melanin to cross the blood-brain barrier. The term melanin derivatives is also intended to include chemical derivatives of melanin, such as an esterified melanin. Melanin Variant: Melanin variants include various subsets of melanin substances that occur as families of related materials. Included in these subsets, but not limited thereto, are:

(1) Naturally occurring melanins produced by whole cells that vary in their chemical and physical characteristics; (2) Enzymatically produced melanins prepared from a variety of precursor substrates under diverse reaction conditions;

(3) Melanin analogs in which a structural feature that occurs in (1) or (2) above is replaced by an unusual substituent divergent from the traditional; and

(4) Melanin derivatives in which a substituent in a melanin produced in (1), (2) or (3) above is further altered by chemical or enzymatic means.

Tyrosinase: An enzyme which, in mammals, catalyzes: (a) the hydroxylation of tyrosine to dopa (3,4-dihydroxyphenylalanine); (b) the oxidation of dopa to dopaquinone; and (c) may catalyze the oxidation of 5,6-dihydroxyindole to indole-5,6- quinone. All of these reactions catalyzed by tyrosinase take place in the biosynthetic pathway which produces melanin. Tyrosinase is most commonly found in a glycosylated form in vivo.

Melanin Concentrating Hormone: Melanin concentrating hormone (MCH) is

a peptide which has been isolated from fish pituitary glands, characterized and synthesized

(Kawauchi, H. et al. , Nature 305, 321 (1983)). MCH has also been localized by

immunohistochemistry in the brain and pituitary gland of salmon, frogs and rats (Baker, B. J. et al. , Gen. Comp. Endocrinol. 50, 1423 (1983), Naito, N. et al. , Neurosci. Lett. 70, 81

(1986), Skotfitsch, G. et al., Proc. Natl. Acad. Sci. USA 83, 1528 (1986) and Zamir, N. et

al., Brain Research 373, 240 (1986)).

A mammalian MCH-like substance has been detected using salmon MCH antiserum directed against salmon MCH by radioimmunoassay and immunohistochemistry (Zamir, N. et al., Proc. Natl. Acad. Sci. USA, supra). This mammalian MCH exhibits distinct chromatographic properties on both Reversed Phase High Performance Liquid Chromatography (RP-HPLC) and gel chromatography when compared to the fish enzyme. Id. The persistence of this mammalian MCH in the mammalian hypothalamo- neurohypophyseal system suggests a role for MCH in posterior pituitary function, such as the regulation of food and water intake. Id.

Other functions of this mammalian MCH peptide have also been suggested. For example, due to the identification of MCH fibers in the human median eminence and pituitary stalk, it has been suggested that the peptide causes the aggregation or concentration of melanin in cells of the central nervous system and may be involved in the regulation of anterior pituitary function (Pelletier, G. et al., Brain Research 423, 247 (1987)). Furthermore, Sekiya, K. et al. (Neuroscience 25, 925, 1988) suggest that MCH may act as a neurotransmitter and/or neuromodulator in the central nervous system or may regulate the pituitary portal-blood system and/or the neurosecretory system in mammals. Melanin: Naturally occurring melanins include eumelanins, phaeomelanins, neuromelanins and allomelanins. Trichochromes which are low molecular weight polymers

derived from the oxidation of tyrosine are also considered melanins for the purpose of this

invention. Melanins and melanin variants are as defined above. Melanin variants are considered melanins for the purpose of this invention unless the context indicates otherwise. The biosynthetic pathway by which melanin is produced is described below as reported by Hearing, V. J. et al., Int. J. Biochem. 19, 1141 (1987).

Naturally occurring melanin is formed through biochemical pathways which involve the hydroxylation and decarboxylation of the amino acids phenylalanine and tyrosine. In one proposed anabolic pathway, tyrosine is hydroxylated to form the catecholamine dopa (3,4-dihydroxyphenylalanine), then the diol is oxidized to form the diketone 3,4-dioxyphenylalanine (also known as dopaquinone). The dopaquinone is cyclized to form 5,6-indolequinone, and the polymerization of those indolequinones produces melanin. There are alternative pathways for melanin production. However, in each of those alternatives an understanding of the mechanisms in the final steps remains elusive.

Naturally occurring melanin can be prepared synthetically or isolated from natural sources. Natural sources include beef eyes, squid, hair, bacteria such as Streptococcus antibioticus , and brain, among others. Melanins can be prepared synthetically, as described by Froncisz, W. et al. (Arch. Biochem. Biophys. 202, 289,

1980) and Lyden, A. et al. (Arch. Int. Pharmacodyn. 259, 230, 1982), among others.

Alternative sources of melanin for use in the present invention are as follows: Leong et al. (U.S. Patent No. 4,855,144, issued August 8, 1989) describe synthetic melanin aggregates which were said to have enhanced biological and physical properties. della-Cioppa et al. (U.S. Patent No. 5,817,585, issued November 15, 1998) describe the production of melanin from transformed E. coli. Melanins, their precursors and their analogs were said to be produced in amounts greater than 3.3 grams wet weight per liter of growth medium. Berliner et al. (U.S. Patent No. 5,726,968, issued July 7, 1998) describe the isolation and purification of melanin from bacteria (Streptococcus antibiolicus) and from beef eyes.

Additional methods for the isolation of melanin are described in Prota, G. , Melanins and Melanogenesis, New York, Academy Press, 1992.

Melanin for use in the present invention is commercially available in powder form from Sigma Chemical Co. (St. Louis, MO), as synthetic (catalog number M8631 and M0418) or isolated from Sepia (catalog number M2649) Therapy

Since the treatment of wounds will usually require many separate doses of melanin and related substances, some regimens will be more preferred than others depending upon the nature of the wound and location. Typical doses would range broadly between about 1 % and about 100% of the wound surface area and preferably between about 5% and about 50% of the wound surface and broadly between about 1 % and about 25% of the volume for internal organ wounds and preferably between about 3% and about 10% of the volume for internal organ wounds.

Melanin in the form of a powder can also be directly applied to the wound or burn.

Topically applied melanin and related substances may be administered once or twice per day for the first week of treatment and then once per day thereafter.

An alternative method for treating wounds with melanin is to enhance the in vivo production of melanin by administering tyrosinase to the patient. Tyrosinase catalyzes at least two, and possibly three, of the reactions in the biosynthetic pathway which produces melanin. Naturally occurring tyrosine in the human body is hydroxylated to 3,4- dihydroxyphenylalanine (dopa), and the hydroxylation is catalyzed by tyrosinase. Tyrosinase also catalyzes the subsequent oxidation of dopa to dopaquinone. The dopaquinone is a precursor for two separate biosynthetic pathways for the production of melanin. Therefore, both tyrosinase-catalyzed reactions which lead to the production of dopaquinone (the hydroxylation of tyrosine to dopa and the oxidation of dopa to dopaquinone) are important reactions in the human body's production of melanin. One pathway from dopaquinone to melanin involves a ring closure and hydrogenation of dopaquinone to produce leucodopachrome. This is followed by partial oxidation of leucodopachrome to dopachrome, and decarboxylation and hydroxylation of the dopachrome to 5,6-dihydroxyindole. The 5,6-dihydroxyindole is then oxidized to indole-5,6-quinone, and it is at this step that tyrosinase is again believed to serve as a catalyst. Korner, A. M. et al., Science 217, 1163 (1982). Tyrosinase is believed to catalyze this oxidation reaction. The indole-5,6-quinone is then converted to melanin or eumelanin.

The other pathway from dopaquinone to melanin involves the addition of cysteine to dopaquinone to produce 5-S-cysteinyldopa, followed by the oxidation of 5-S- cysteinyldopa to 5-S-cysteinyldopaquinone. A ring closure of the 5-S-cysteinyldopaquinone then yields 7-alanyl-5-hydroxy-3-carboxy-2H-l,4-benzothiazine which is subsequently decarboxylated to yield 7-alanyl-5-hydroxy-2H-l,4-benzothiazine. At this point, the 7- alanyl-5-hydroxy-2H-l,4-benzothiazine is converted to melanin and pheomelanin. Tyrosinase does not play any additional role in this melanin production pathway.

The tyrosinase can be administered by any means which will insure that it reaches the desired tissue. Several routes of administration can be used such as topical and parenteral, e.g. directly into the wound. Since the treatment will require many separate doses of tyrosinase, and some mechanisms will be more preferred than others. The amount of tyrosinase administered must be sufficient to catalyze the melanin-producing reactions such that sufficient melanin is produced to treat or prevent the wound. Proper doses are those sufficient to result in production of melanin in amounts described above as % surface area or % volume and can be determined using routine experimental known to those of ordinary skill in the art. Another method by which the in vivo production of melanin may be enhanced is by the administration of the tyrosinase gene to the effected patient. After administration, the tyrosinase gene is transcribed and translated and tyrosinase is produced. The tyrosinase, in turn, catalyzes the production of melanin from naturally occurring melanin precursors as explained above.

Tyrosinase Gene The gene for human tyrosinase has been isolated, sequenced and cloned (PCT application WO 88/02372, published Apr. 7, 1988). The cloned gene encodes a polypeptide of 548 amino acids with a molecular weight of 62, 160, excluding a hydrophobic signal peptide. The gene for Streptomyces glaucescens tyrosinase has also been isolated and sequenced (Huber, M. et al., Biochemistry 24, 6038 (1985)). Nearly all of the codons used end in either G or C, and the overall G+C content of the gene is 71.4% . Id.

The S. glaucescens tyrosinase gene may be isolated as follows. The Kpnl fragment of plasmid pMEA4 containing the S. glaucescens gene (Hintermann, G. et al., Mol. Gen. Genet. 200, 422 (1985)) is cloned into the PvuII site of pBR322 with Kpnl linkers (P-L Biochemicals). Two resulting plasmids (pMEA6 and pMEA7) contain the tyrosinase gene in opposite directions. (Huber, M. et al. , supra). Plasmid DNA is then isolated by conventional techniques such as those described by Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor (1982). Restriction endonucleases are then used according to the suppliers' instructions (Boehringer, Amsterdam) to perform digestions, and the fragments are recovered by low-melting agarose gels as described by Weislander, L., Anal. Biochem. 98, 305 (1979). The nucleotide sequences are then determined using methods well known to those of ordinary skill in the art. The tyrosinase gene can be introduced into the mammalian system by its incorporation into a vector. The amount of tyrosinase gene administered must be sufficient to transfect susceptible mammalian cells so that tyrosinase is produced therefrom.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

Vectors typically comprise DNA or RNA of a transmissible agent, into which foreign DNA or RNA is inserted. In the case of DNA, a common way to insert one segment of DNA into another segment of DNA involves the use of restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A "cassette" refers to a coding sequence or segment of DNA or RNA that codes for an expression product that can be inserted into a vector at defined sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, referred to as a "DNA construct." A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Prophylaxis

In another aspect, the present invention is directed to administering an effective amount (as disclosed above) of an active substance such as melanin and related substance or tyrosinase (either the protein or the gene encoding tyrosinase) to prevent the formation of wounds in susceptible patients. As mentioned above, one method by which tyrosinase may be introduced into the mammalian system is by its incorporation into a vector as defined above. In this way, areas of the body which are susceptible to decubitus ulcers and bed sores, such as the back and feet in diabetics can be protected.

Pharmaceutical Compositions and Delivery

Pharmaceutical compositions comprising the active substance of the present invention (i.e. melanin, melanin variant, melanin derivatives, tyrosinase, tyrosinase gene, MCH and combinations thereof) in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the preparation desired for administration, e.g., oral, topical, aerosol, suppository, parenteral or spinal injection. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). If desired, tablets may be sugar-coated or enteric-coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, to aid solubility or for preservative purposes, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, pH adjusting agents, isotonicity adjusting agents and the like may be employed. For topical administration (which is preferred), the carrier may take a wide variety of forms depending on the form of preparation, such as creams, dressings, gels, lotions, ointments or liquids. Aerosols are prepared by dissolving or suspending the active ingredient in a propellant such as ethyl alcohol or in propellant and solvent phase. Suppositories (for treating, e.g. , hemorrhoids) are prepared by mixing the active ingredient with a lipid vehicle such as theobroma oil, caca butter, glycerin, gelatin, or polyoxyethylene glycols. The pharmaceutical compositions for topical or aerosol administration will generally contain from about 1% by weight to about 40% by weight melanin and related substances, depending on the particular form employed. Liquid formulations would broadly content between about 0.01 % and about 50% and preferably about 3% of melanin and related substances. It should be realized that 1 gram of a gel or an ointment will cover about a 6 inch diameter of skin.

Melanin, either natural or synthetic, can be incorporated into substances, mixtures or devices which cover or are implanted into wounds (see Table 1 below). Alternatively, it has been discovered that stimulating increases in the amount of natural melanin will help prevent and treat wounds.

Other methods for increasing the concentrations of melanin and related substances can also be used. For instance in the case of skin wounds, (1) placing a patient in the sun to stimulate melanogenesis, or (2) supplementing copper in the food or parenteral nutrition in a hospitalized or nursing home patient will stimulate melanogenesis. Copper is a critical component of tyrosinase (Prota,G., Melanins And Melanogenesis, New York: Academic Press, 1992), which is a critical enzyme in the melanin synthesis pathway.

In light of the above, copper is another preferred compound to increase melanin synthesis in the body pursuant to the present invention. Agents which affect copper levels in the body (and in specific tissues) thus may also have utility. Some of the substances that cause changes in copper levels include the following: (I) Metals such as zinc, cadmium and iron (Linder and

Hazegh-Azam, Am J. Clin Nutr, 1996, 63:797S-811S);

(II) Enzymes that affect the metabolism of copper and/or

use copper as their cofactor (e.g. , superoxide

dismutase, hyaluronidase); copper binding proteins

(e.g. , ceruloplasmin or metallothiein) (Linder and

Hazegh-Azam, supra);

(III) Substances that affect the synthesis or degradation of copper

binding proteins/enzymes (e.g. , retinoic acid [Song and

Levenson, Int. J. Vitam. Nutr. Res. , 1997, 67: 141-144]);

(IV) Decreasing Copper binding non-proteinaceous compounds

(e.g. , ascorbate [Itoh and Eguchi, Dev. Biol. , 1986, 115:353-

362; Droudin et al. , Eree Radic Biol Med, 1996, 21:261-273],

various thioureas [Masuda and Εguchi, Cell Structure and

Function, 1984, 9:25-35], guanosine, adenosine

[Masuda and Εguchi, Inorganic Chemistry, 1990, 29:3631],

cytosine [Palaniandavar et al. , J. Chemical Soc , 1996,

7:1333] and their phosphates).

(V) Diet: The richest dietary source of copper are animal livers,

Crustacea, shell fish, dried fruit, nuts and chocolate (Excerpta

Medica 1980 Ciba Foundation Symposium 79, p.5-22;

Olivares, M and Uauy, B. Am. J. Clin.Nut. 1996, 63:7915- 7965). The following are examples of the range of formulations of melanin for preventing

or treating wounds.

1. Natural or synthetic melanins as powders, beads, particles, strips, patches, nets or matrices. 2. Any of the above can be enclosed in a packaging layer or layers such as in liposomes.

3. Melanin can be incorporated as melanosomes, such as can be isolated by various sorting technologies such as differential centrifugation of melanocytes.

4. Melanin can be incorporated in any of the above formulations into nonliving materials (organic or no-organic) for uses as a patch, paste, gel, cream, ointment, mixture, plug or implant. A list of some of the types of nonliving materials into which melanin can be incorporated is set forth in Example 1 below.

5. Melanin can also be incorporated into living materials, such as commercially available epidermal, dermal, or epidermal-dermal skin replacements and patches.

6. Melanin can be used along with other substances that either add or synergize with the beneficial properties listed above.

Melanin is insoluble in aqueous media. Liquid formulations would be in the form of a suspension which could be agitated before use. Because melanins are highly conserved in evolution, without wishing to be bound by theory, it is believed that the use of melanins from synthetic sources or lower animals is unlikely to provoke a deleterious immune response. Furthermore, should autologous melanin be required or preferred, cells containing the subject's own melanin can be easily isolated from their skin, and reproduced in vitro. (Prota, G. Melanin and Melanogenesis, NY Academy Press 1992.)

Because melanin is paramagnetic, electrical and magnetic means could be used to adjust the arrangement of melanin in melanin containing wound healing devices, such as those described above.

It is believed that the methods described above will be particularly useful in creating materials to prevent and treat decubitus ulcers (bedsores). Decubitus ulcers are a source of much misery for the human race. Patients who are immobile or debilitated for any reason including trauma, surgery, age or incapacity are greatly at risk for these painful wounds which can occur in a matter of hours and can easily become infected. Furthermore such wounds can lead to large areas of the body, superficial or deep becoming necrotic, requiring removal or amputation, and the infections and morbidity resulting from such wounds can lead to death (Parish, L.C. et al. , (Eds.) The Decubitus Ulcer In Clinical practice, New York: Springer 1997).

Decubitus ulcers tend to develop from pressure, abrasive and shearing forces from movement, leading ultimately to compromise of the vascular system and death of skin, subcutaneous tissue and muscle, accompanied by infection (Parish,L.C.,Witkowski et al. (Eds.) The Decubitus Ulcer In Clinical practice, New York: Springer 1997).

It has been discovered that melanin and related substances and preparations incorporating such materials in the manners listed above, are ideally suited to prevent and treat wounds such as decubitus ulcers . More specifically, the hardness of melanin resists pressure. Its packaging into small particles or melanosomes separated by flexible membranes (or living or nonliving softer substances) permits flexibility to accommodate to the curvature of the appropriate body area. Its properties of being anti-inflammatory and anti-infectious prevent further breakdown and pathogenic microorganism invasion of the area at risk. Melanin's ability to bind toxins helps absorb toxins produced by microorganisms and other foreign substances that contaminate the wound. Melanin can also provide a substrate along which cells can move thus enhancing cell migration and healing. It remains superficial, so that it can be cast off or removed once the wound is sealed. In addition, it also forms a physical barrier to any additional noxious agents. Wounds of all types, including decubitus ulcers, burns and traumatic injuries, result in discontinuities in one or more tissues comprising an organ. The capacity of the cells in a tissue to proliferate spontaneously in response to a wound in a tissue, is a characteristic of most cell types. This capacity is independent of vascularization and can be demonstrated for most cells in vitro, with no blood supply. For example, an epithelium is generally a layered population of contiguous cells of the same type in which a germinative population sits on a basement membrane and gives rise to progeny which migrate away from the direction of the basement membrane. e.g. skin, intestinal epithelium, urinary bladder epithelium, etc. All of these epithelia are avascular, with nutrient and waste exchange occurring without a blood supply within the epithelial.

In addition, the skin is composed of the following layers: stratum germinativum, statum spinosum, stratum granulosum, stratum corneum. No blood supply exists within any of these layers. A wound results in the proliferation of the basal cells and the resulting cells migrate to cover the discontinuity and reestablish a barrier to the outside environment. A similar situation occurs in other epithelia when wounded. It also occurs in other cell types such as fibroblasts in connective tissue, renal parenchyma, etc. Without wishing to be bound by theory, it is believed that melanin, when supplied in a non-living matrix material e.g., silicone patch or a living cellular product, such as a melanized version of the commercial product Apligraf, (available from Organogenesis, Inc., Canton, MA) facilitates healing by providing a hard but flexible barrier to the outside environment under which normal proliferation and reparative processes (such as those intrinsic to avascular epithelia, as discussed above) can occur . Melanin makes a patch layered on the skin hard and thus more able to resist pressure which would otherwise damage the epithelium and compromise the underlying blood supply. Melanin also helps maintain homeostasis by enhancing the barrier function of skin by being hard, binding potential environmental toxins, limiting transcutaneous water loss, and by its antimicrobial properties. This is totally independent of any affect on angiogenesis.

Melanin helps prevent a wound, (in cases where the melanization of the tissue e.g. the stratum corneum is enhanced by hormonal treatment or enzyme, e.g., tyrosinase enhancement) by permitting individual cells and cell products (such as the nonliving keratin sheet which the stratum corneum represents) to resist pressure, infectious agents, and toxins. Thus it is proposed that because of its hardness and other properties, melanin will make a stronger barrier enabling normal skin cells to better resist wounding and to repair wounds. This is totally independent of any affect on angiogenesis.

In alternate preferred embodiments of the present invention, amounts of melanin and related substances effective to treat wounds in mammals can be incorporated into drugs (such as antibiotics) which are topically applied to wounds. Since skin cells naturally take up melanin and related substances (by pinocytosis or phagocytosis), the incorporation of melanin and related substances into commercially available topical antibiotic and antifungal creams and ointments will allow melanin to be taken up by cells in the wound this will have the effect of strengthening the wound and sealing it to prevent further infection. In this embodiment, melanin and related substances may be incorporated into topical or injectable medications at a concentration broadly ranging between about 0.5% and about 3 % by weight and preferably 0.5% by weight. Non-limiting examples of suitable topical antibodies include bacitracin, polymyxin and muporicin (the latter available as a cream or ointment from SmithKline Beechman, Philadelphia, PA.)

Any topical medication applied to the wound, such as topically applied growth factors (e.g. Regranex by Johnson and Johnson), or emollients such as petrolatum or aquafor, or mineral oil, which are themselves believed to enhance wound healing by topical application can be used with effective amounts of melanin and related substances described above.

Presented below are general categories, and specific examples as to topically applied substances which can be used. These substances can be uses alone or in any combination. These material are available from numerous commercial sources, such as Sigma Chemical Co. or Fisher Scientific.

AUXILIARY EMULSIFYING AGENTS/EMULSION STABILIZERS

Lanolin and Lanolin derivatives

STABILIZERS (including preservatives, antioxidants and chelating agents)

Glycerin

HUMECTANTS Sorbitol solutions EMULSIFYING AGENTS Sodium Lauryl Sulfate EMOLLIENTS Mineral Oil SOLVENTS Propylene glycol THICKENING AGENTS Petrolatum The present invention is described below in examples which are intended to further describe the invention without limiting its scope.

Example 1. Melanin-Containing Materials.

Presented below in Table 1 is a non-limiting list of commercially available wound dressing which can be impregnated with melanin and related substances.

Table 1. Examples of moisture-retentive dressings and manufacturers in the United

States

The pharmaceutical formulations and melanin containing devices listed above need not contain effective amounts (as defined herein) of melanin and related substances since such effective amounts can be reached by administering a plurality of such materials. Effective amounts of melanin and related substances can be determined as follows:

1. Physical Tests:

(a) Physical tests of wound dressings showing that melanin

enhances their hardness, ability to resist abrasion, barrier

functions, and ability to immobilize toxins;

(b) Physical tests of pharmaceutical preparations showing that

melanin enhances the skin's (or other organ's) hardness, ability to resist abrasion, barrier functions, and ability to

immobilize toxins, especially at wound sites.

2. Animal Tests:

(a) Tests on animal models showing that melanin in wound

dressings enhances the rate of wound healing, and imparts to

the wound site an anti-inflammatory and anti-infective

environment;

(b) Tests on animal models showing that melanin in

pharmaceutical_preparations enhances the rate of wound

healing, and imparts to the wound site an anti-inflammatory

and anti infective environment.

3. Human Clinical Tests:

Human clinical studies, showing (1) and (2) above apply to human

wound healing and prevention of wounds.

Example 2 Presented below are non limiting categories of topical agents and specific examples of commercially available products into which melanin and related substance may be incorporated.

Burn Treatments

Silver Sulfadiazine Cream 1 % (catalog number. 32886 Henry Schein, Inc.)

Emollients

Ammonium Lactate Cream (catalog number. 1027036, Henry Schein, Inc.)

Ichthammol Ointment - 20% (catalog number 1020956, Henry Schein, Inc.) Lanolin (catalog number 1021756, Henry Schein, Inc.

Sun Screen Products

Zinc Oxide and ointment (catalog number 4711456, Henry Schein, Inc.)

Antibiotics and Antibacterials

Bacitracin Ointment (catalog number 4706972, Henry Schein, Inc.)

Clindamycin Topical solution (catalog number 1028791, Henry Schein, Inc.)

Clotrimazole Cream (catalog number 1027125, Henry Schein, Inc.)

Erythromycin topical solution (catalog number 4207358, Henry Schein, Inc.) Gentamycin ointment (catalog number 4733872, Henry Schein, Inc.)

Nystatin cream (catalog number 4201056, Henry Schein, Inc.)

Antifυngals and Antiseborreics

Clotrimazole Betamethasone cream (catalog number 1025190, Henry Schein, Inc.)

Ketoconoizole Cream (catalog number 1024347, Henry Schein, Inc.) Miconiazole Nitrate Cream (catalog number 2723761, Henry Schein, Inc.)

Nystatin Ointment (catalog number 1020986, Henry Schein, Inc.)

Antiseptics

Alcohol, Isopropyl 91 % (catalog number 1020810, Henry Schein, Inc.)

Hydrogen Peroxide (catalog number 1023516, Henry Schein, Inc.)

Isopropyl alcohol 70% (catalog number 1024716, Henry Schein, Inc.)

Povidone Ointment (catalog number 4722656, Henry Schein, Inc.)

Claims

What is Claimed is: 1. A method for treating a wound in a mammal comprising administering to a mammal in need of such treatment an amount effective for treating said wound of an agent which causes an increased concentration of melanin and related substance in said wound.

2. The method of claim 1 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

3. The method of claim 2 wherein said wound is a decubitus ulcer.

4. The method of claim 2 wherein said administration is selected from oral, parenteral, topical and by aerosol.

5. The method of claim 3 wherein said administration is topical.

6. A method for preventing a wound in a mammal comprising administering to a mammal in need of such treatment an amount of an agent effective to prevent said wound of an agent which causes an increased concentration of melanin and related substances in said wound.

7. The method of claim 6 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

8. The method of claim 6 wherein said administration is topical.

9. The method of claim 8 wherein said agent is tyrosinase or a gene encoding tyrosinase.

10. The method of claim 6 wherein said wound is a decubitis ulcer.

11. The method of claim 6 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

12. A wound healing device selected from a patch, a strip, a wound dressing or a band comprising an amount of melanin or related substance effective to treat a wound in a mammal.

13. The wound healing device of claim 12 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

14. A pharmaceutical formulation for treating wounds in a mammal comprising mineral oil and a wound healing effective amount of an agent which causes an increase in the concentration of melanin in said wound.

15. The pharmaceutical formulation of claim 14 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixmres thereof.

16. A pharmaceutical formulation for treating wounds in a mammal comprising a topical antibacterial cream or ointment and a wound healing effective amount of an agent which causes an increased concentration of melanin and related substances in said wound.

17. The pharmaceutical formulation of claim 16 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixmres thereof.

18. A method for treating wounds in a mammal comprising administering to a mammal in need of such treatment an amount effective to treat said wound of a pharmaceutical formulation of claim 14 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixmres thereof.

19. The method of 18 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixtures thereof.

20. A pharmaceutical formulation for treating wounds in a mammal comprising an emollient selected from ammonium lactate, ichthammol ointment and lanolin and a wound healing effective amount of an agent which causes an increased concentration of melanin and related substances in said wound.

21. The pharmaceutical formulation of claim 20 wherein said agent is selected from melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, tyrosinase, tyrosinase gene, melanin concentration hormone and mixmres thereof.

22. The pharmaceutical formulation of claims 14, 15 or 20 further comprising a pharmaceutical acceptable carrier or diluent.